Purinergic Calcium Responses in Human Müller Cells

Abstract

Purpose : We have previously shown purinergic P2X₇ receptor –mediated retinal ganglion cell (RGC) death in the human retina. Müller cells are the major retinal glial cell and a possible contributor. The purpose of this research was to characterise the P2X₇ and other purinergic (P2) receptor calcium responses in the human Müller cell.

Methods : The immortalised human Müller cell line MIO-M1 was used. Initial experiments investigated cell viability and death in response to the purine agonists ATP and BzATP across 24h. Cell viability and death were evaluated using MTS and LDH assays respectively. Fura-2 assay was used to measure intracellular calcium changes in response to ATP and BzATP in MIO-M1 cells.

Results : There was no significant change in cell viability or death when stimulated with ATP (100-3000µM) and BzATP (10-300µM) (n=4). ATP (0.01-1000µM) caused a dose-dependent increase in intracellular calcium concentration with an EC₅₀ of 0.7µM (n=4). BzATP (10-1000µM) caused a dose-dependent increase in intracellular calcium concentration with an EC₅₀ of 57.3µM (n=4). The selective non-competitive P2X₇ antagonist AZ 10606120 (10µM) entirely inhibited the BzATP-calcium response at all concentrations (p<0.05) (n=4). AZ 10606120 (10µM) partially inhibited the ATP-calcium response at the highest concentration of 1000µM only (p<0.05) (n=4). The P2Y₁ antagonist MRS 2179 (10µM) partially inhibited the ATP-calcium response at concentrations <100µM (p<0.05) (n=4). Selective purinergic antagonists AR-C 118925XX (P2Y₂) and MRS 2578 (P2Y₆) showed no significant antagonism of the ATP-calcium response (n=4).

Conclusions : This data showed functional evidence of the P2X₇ receptor in MIO-M1 cells. BzATP induced calcium responses were entirely P2X₇-mediated; ATP only produced a P2X₇-mediated calcium response at higher concentrations, in keeping with these concentrations acting as a danger signal via this receptor. The remainder of ATP-induced calcium responses could be attributed to P2Y₁ and other purinergic receptors which mediate homeostatic functions in the retina. Purinergic agonists at concentrations demonstrated to be stimulating the P2X₇ receptor did not result in P2X₇-mediated cell death.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.